Aims: To investigate the role of β-adrenoceptor subtypes in mediating relaxation of the pig bladder trigonal muscle in vitro. Materials and Methods: Longitudinal strips of trigonal muscle were isolated, and the mucosa and serosa removed. Tissues were precontracted with KCl, and β-adrenoceptor agonists (isoprenaline or salbutamol) were added cumulatively, and concentration-relaxation curves (CRCs) were obtained. CRCs to agonists were obtained in the absence and presence of antagonists and antagonist affinity values were calculated. Results: Isoprenaline (non-selective β-agonist) relaxed with high potency (pEC50 = 7.2). Propranolol antagonized CRCs to isoprenaline with a high affinity (apparent pKB = 8.8), but the Schild plot had a slope significantly (P < 0.01) less than unity (0.61), suggesting that responses were mediated by more than one β-adrenoceptor subtype. CGP20712A (β1-antagonist) antagonized responses to isoprenaline with a low affinity (apparent pKB = 5.13), indicating β1-adrenoceptors did not participate in the response. The affinity of ICI118551 (β2-antagonist) for antagonism of responses to isoprenaline was also relatively low (apparent pKB = 6.9), and the Schild slopes were significantly (P < 0.01) less than unity (0.58). SR59230A (β3-antagonist) antagonized CRCs to isoprenaline with a relatively low affinity (apparent pKB = 7.5), and with a Schild slope significantly (P < 0.01) less than unity (0.86), indicating that responses may be mediated by more than one β-adrenoceptor subtype. In contrast to that observed with isoprenaline, the β2-adrenoceptor selective agonist salbutamol induced relaxation with a relatively low potency (pEC50 = 6.6) and with maximum responses of 80% of that to isoprenaline. ICI118551 competitively antagonized (Schild plot of unity) responses to salbutamol with a high affinity (pA2 = 8.3). Conclusions: These data suggest that β-adrenoceptor mediated responses to isoprenaline of the bladder trigone are mediated via both the β2- and β3-adrenoceptor subtypes whilst responses to salbutamol appear to be mediated only via the β2-adrenoceptor.